Although it is clear that specific transport systems in the plasma membrane are responsible for regulation on intracellular levels of essential nutrients, very little is known about transport processes at a molecular level. We plan to develop well-defined systems for studying transport in mammalian cells and intend to use both genetic and biochemical approaches to these problems by examining transport of purines by cells growing in tissue culture. The mechanism of hypoxanthine and quanine transport by Chinese hamster fibroblasts is to be examined by both kinetic and metabolic studies. A group of 8- azaguanine-resistant (8-AGr) mutants will be used in these experiments. Some of the 8-AGr mutants are defective in hypoxanthine guanine phosphoribosyltransferase (HGPRT), while others have normal levels of HGPRT. The former class of mutants will be used for study of purine transport in the absence of subsequent metabolism, while the latter class of CHL cells seems likely to contain guanine transport mutants that will be useful for defining the membrane components involved in purine transport. We plan also to develop methods for the isolation of mammalian plasma membrane vesicles that retain transport capacity. This in vitro system will be applied to study of hypoxanthine and guanine transport in membrane vesicles isolated from wild-type and 8-AGr clones of Chinese hamster fibroblasts. These experiments will focus on the regulatory mechanisms governing purine transport.